Combinatorial approaches to protein folding and function

描述：（由申请人提供）蛋白质的折叠和稳定性具有 成为从分子水平理解癌症等疾病的关键因素 和老年痴呆症然而，对这些现象的完整理解仍然存在 难以捉摸一个非常有效的方法是系统地重新设计 proteins.重新设计的一个重要限制是 有大量的可能变异可以被设计出来。的方法 对大量已被充分研究的蛋白质变体文库的分析是非常重要的。 令人向往蛋白质折叠和稳定性的一个非常好的研究模型是 四螺旋束蛋白Rop，其调节科尔1质粒拷贝数。 这将作为Rop功能屏幕的基础，其中 从科尔1质粒生产标记物样绿色荧光蛋白 反映了Rop蛋白变体的完整性。这样的屏幕将用于 从含有详尽的Rop变体的大型文库中鉴定活性Rop变体 环或核心残基中的突变。一个有趣的问题是 识别控制整体构象的参数。最近 实验表明，蛋白质可以折叠成高度不同的三维结构， 尽管同源性高，但结构（例如， α/β B1结构域折叠成Rop的卷曲螺旋结构）。绳状 结构已经通过金属离子通过新的工程化来稳定， 金属结合位点我们将通过以下方式来研究构象转换的极限： 使用金属结合来稳定来自B1变体的Rop样结构。我们 也对分离再现Rop功能的B1变体感兴趣。一 筛选Rop功能将使我们能够从根本上扩大蛋白质的范围， 重新设计和完善我们对控制蛋白质的参数的理解 折叠和稳定性。

DESCRIPTION: (provided by applicant) Folding and stability of proteins have emerged as key elements in the molecular understanding of diseases like cancer and Alzheimer?s, yet a complete understanding of these phenomena remains elusive. One highly productive approach has been the systematic redesign of proteins. A significant limitation to redesign is the characterization of the enormous number of possible variants that can be engineered. Methods for the analysis of large libraries of variants of well-studied proteins are highly desirable. An extremely well-studied model for protein folding and stability is the four-helix bundle protein Rop, which modulates ColE 1 plasmid copy number. This will serve as the basis for a screen for Rop function, wherein the production of a marker like green fluorescent protein from a ColE 1 plasmid reflects the integrity of a Rop protein variant. Such a screen will be used to identify active Rop variants from large libraries containing exhaustive mutations in loop or core residues. One interesting problem is the identification of parameters that control overall conformation. Recent experiments show that proteins can fold into highly disparate threedimensional structures despite high homology (for example, a variant of the alpha/beta B1 domain folds into the coiled-coil structure of Rop). Rop-like structures have been stabilized by metal ions through the engineering of novel metal-binding sites. We will examine the limits of conformational switching by using metal binding to stabilize a Rop-like structure from a B1 variant. We are also interested in isolating B1 variants that recapitulate Rop function. A screen for Rop function will allow us to radically expand the scope of protein redesign and refine our understanding of the parameters that control protein folding and stability.